Detailed protocols for all the experiments we developed can be found at the end of this page. To better understand the rationale behind our experimental work, please take a look here.

Bacteria

Qualitative assays for silicate solubilization

Two approaches were used to qualitatively confirm the silicate solubilizing activity of Pseudomonas fluorescens:

The first method involved growing the bacteria on solid growth media supplemented with 0.25% calcium aluminosilicate and subsequently checking for clearance zones around the colonies, which are indicative of solubilization ^[1]^[2].
The second method involved using NBRISSM, a defined differential liquid media for screening silicate solubilizers ^[3]. Every 1 liter of NBRISSM used in our experiment contained: 2.5 gm glucose; 2.5 gm hydroxyapatite; 1.25 gm MgNO₃; 1.25 gm CaCl₂; 0.1 gm (NH₄)₂SO₄; 0.1 gm calcium aluminosilicate in distilled water. We also added 0.75% bromocresol purple (BCP) pH indicator to reflect the presence of silicic acid through a change in color from purple to yellow.

See our results here.
Learn more about our DBTL cycles here.

Growth of bacteria in Martian soil simulant

The Mars Global (MGS-1) High-Fidelity Martian Dust Simulant ^[4] was obtained from Prof. Sathyan Subbaiah, Department of Mechanical Engineering, IIT Madras.
To inoculate the soil, a 5 ml overnight culture of P. fluorescens was centrifuged to collect the cells. The pelleted cells were resuspended in 5 ml of fresh LB media.
This cell suspension with fresh growth media was poured into 15 gm of Martian soil simulant (1:3 mass ratio) to achieve a moisture level of 25%.
The flask with inoculated soil was placed in a 30℃, 250 rpm shaking incubator.
Daily, over a period of 10 days, 1 gm of soil was mixed with 9 ml of sterile water and centrifuged. The supernatant was then serially diluted and plated on LB plates.
The growth of the bacteria in soil was observed by counting the CFU (colony forming units) every day.

See our results here.
Learn more about our DBTL cycles here.

Quantitative assay for silicate solubilization in soil

After the bacteria was grown in soil, the solubilized silicates were extracted from soil using calcium chloride. 0.01 M CaCl₂ and soil in the ratio (10:1) were taken in a tube and continuously shaken for 1 hour ^[5].
After shaking, the tube was centrifuged at 6000 rpm for 2 minutes. The supernatant was filtered and used for quantification of solubilized silicates using the molybdenum blue method.
0.25 ml of filtrate was taken in a plastic centrifuge along with 10.5 ml of distilled water, 0.25 ml of 1:1 hydrochloric acid and 0.5 ml of 10% ammonium molybdate.
After 5 minutes, 0.5 ml of 20% tartaric acid was added.
After 2 minutes, 0.5 ml of a solution of the reducing agent 1-aminonaphthol-4-sulfonic acid (ANSA) was added. To prepare the ANSA solution, (a) 1.6 gm ANSA and 8.0 gm of sodium sulfite were added in 100 mL water, and (b) 100 gm sodium bisulfite was added to 800 ml water. The solutions (a) and (b) were mixed and the volume was made to 1 liter to obtain the final ANSA solution ^[6].
After 5, but no later than 30 minutes following addition of ANSA, the absorbance was measured at 630 nm using a UV-Vis spectrophotometer.
Simultaneously, silica standards of 0.2, 0.4, 0.8, and 1.2 mg L^-1 were prepared in the same sodium sulfite matrix and had their absorbances measured using the spectrophotometer ^[7].

Learn more about our results and DBTL cycles here.

Preparation of BioBricks

After locating the destination vector, pJUMP24-1A(sfGFP), and required parts in the iGEM Distribution Kit 2024, the following steps were performed:

The dried down DNA in the wells was suspended in 10 μl demineralized water.
1 μl of this mixture was used to transform competent E. coli DH5α cells. Aliquots of competent cells were thawed on ice and then mixed with DNA. The cells were given a heat shock for 30 seconds at 42℃, followed by a cold shock for 2 minutes. They were then immediately suspended in 1 ml of SOC (enriched media) for recovery and incubated in a 37℃ shaking incubator for 90-120 minutes.

Part Name	Part Type	Vector Backbone in Distribution Kit	Antibiotic Used
BBa_J23100	Promoter	pSB1C5SA	Chloramphenicol 35 µg/ml
BBa_J428038	Ribosome Binding Site	pSB1C5SB	Chloramphenicol 35 µg/ml
4S-limonene synthase	Coding Sequence	BBa_J435500	Ampicillin 100 µg/ml
BBa_J428092	Terminator	pSB1C5SD	Chloramphenicol 35 µg/ml

The cells were then plated on appropriate antibiotic plates.

Transformed colonies were picked and inoculated in LB media with appropriate antibiotics.
The overnight culture was then used to isolate plasmids following the QIAprep® Spin Miniprep Kit protocol.
Concentrations of isolated plasmids were measured using a Nanodrop spectrophotometer.

Learn more about our results and DBTL cycles here.

Golden Gate Assembly

The assembly protocol recommended by iGEM Technology was used ^[8].
We used the NEB Calculator to compute the mass of each BioBrick to be added to the reaction mixture in order to achieve 25 fmol of each BioBrick and 12.5 fmol of the destination vector.
A 10 μl reaction was set up with 1 μl of BsaI, 0.5 μl of T4 DNA ligase, 1 μl ligase buffer and the computed amounts of DNA. The rest of the reaction mixture consisted of deionised water.
The following thermal cycling protocol was then followed:

1 cycle	37°C	10 mins
15 cycles	37°C	1.5 min
15 cycles	16°C	3 mins
1 cycle	50°C	10 min
1 cycle	65°C	10 min

The reaction mixture was then mixed at 10% volume with 50 μl competent E. coli DH5α cells.
Transformation through heat shock was carried out and the cells were plated on kanamycin (35 μg/ml) plates.
Positive colonies were picked and propagated in antibiotic media. The culture was then used for plasmid isolation.

See our results here.
Learn more about our DBTL cycles here.

Confirmation of plasmid assembly

After Golden Gate Assembly and E. coli plasmid isolation, the plasmid was subjected to a single digestion by XbaI and a double digestion by XbaI and BstbI.
The cloning vector without our cassette was also digested by XbaI.
A 25 μl reaction was set up with 2.5 μl buffer, 0.5 ml of each enzyme, 500 ng of plasmid DNA and deionized water.
Following digestion at 37℃ for 4 hours, we loaded the three digestion products in 0.8% agarose gel. The electrophoresis was run for 30 minutes at 100 volts. Gel was then viewed under UV Transilluminator.

See our results here.
Learn more about our DBTL cycles here.

Transformation of P. fluorescens

Competent Pseudomonas fluorescens cells were prepared by washing the cells in late logarithmic phase with MEB (magnesium electroporation buffer). MEB contains 1 mM MgCl₂ and 1 mM HEPES. For the washing step, a cell suspension in MEB was centrifuged at 2300g for 10 minutes at 4℃. The wash step was repeated thrice and all the steps were performed on ice. After all the wash steps were done, the cell pellet was resuspended in 100 μl MEB and aliquoted to make glycerol stocks ^[9]^[10].
Prior to electroporation, 50 μl of cell suspension aliquots (from the glycerol stock) were thawed on ice and mixed with: (a) 150 ng of DNA and (b) 1000 ng of DNA.
Electroporation cuvettes were chilled on ice.
The electroporator was set to 25 µF, 200 Ω, 2.5 kV/cm ^[11].
The mixtures of cells and DNA were then transferred to cold electroporation cuvettes.
Applying a pulse at the mentioned settings produced a time constant of 5.0 - 5.1 ms.
Immediately, the cells were suspended in 1 ml SOC.
This was then carefully transferred to sterile culture tubes and allowed to incubate at 30℃ and 225 rpm.
After incubation, the cell suspension was spread on 25 μg/ml kanamycin plates and placed in a 30℃ incubator.
Positive colonies were screened the next day.

See our results here.
Learn more about our DBTL cycles here.

Diatoms

Culturing diatoms

Phaeodactylum tricornutum SAG 1090-1a was obtained from the Culture Collection of Algae at the Georg-August-University in Göttingen, Germany.
Under normal conditions, the diatoms were cultivated in BG11 medium ^[12] prepared using natural seawater, with pH maintained between 7 and 8. Growth conditions were maintained at either 27°C and 160 rpm or 21°C and 150 rpm, with a light dark cycle of 12h:12h and an average light intensity of 1800 lux.
Stressed diatoms were rescued by supplementing them with a carbon source. The culture was grown in f/2 medium supplemented with glycerol, made to a final concentration of 0.1 M ^[13]. The pH was adjusted to 8.

See our results here.
Learn more about our DBTL cycles here.

Minimal media culturing

Growth conditions of P. tricornutum in a minimal medium were studied. This was done as an effort to make sure that diatoms could be grown with the least amount of resources given the premise of in situ resource utilization.
The diatoms were cultivated in natural seawater supplemented with nitrogen and phosphorus sources ^[14].
Growth conditions were maintained at 27°C and 160 rpm with a light dark cycle of 12h:12h and an average light intensity of 1800 lux.
Growth was quantified by measuring the optical density of the culture at 750 nm, in intervals of 24 hours.

See our results here.
Learn more about our DBTL cycles here.

Effect of silicon on diatom growth

P. tricornutum was cultured separately in minimal medium and minimal medium containing 30 mg/L sodium metasilicate nonahydrate ^[14], starting with the same initial OD of 0.05.
This was carried out by pelleting the diatom cells from the stock culture by centrifugation of equal volumes of the culture.
Growth conditions were maintained at 27°C and 160 rpm.
The experiment was carried out in duplicates and the growth was quantified by measuring OD at intervals of 24 hours for 15 days.

See our results here.
Learn more about our DBTL cycles here.

Preparation of genes for acetaminophen synthesis

We sought out to demonstrate the dual biomanufacturing capabilities of our co-culture system by engineering the diatoms to synthesize acetaminophen.
We received the P. tricornutum-specific plasmids pPTbsr and pPtPuc3 from Addgene as stab cultures. After propagating the culture on appropriate antibiotics we made glycerol stocks and isolated plasmids from the culture.
We decided to insert our genes of interest for acetaminophen synthesis - 4abh and nhoA in the multiple cloning site flanked by the fcpA promoter (a constitutive promoter) and terminator.
Primers were designed for Gibson assembly of the 2 gene sequences.
For PCR, a 10 μl reaction was set up with 2 μl buffer, 0.2 μl dNTPs, 0.1 μl of DNA polymerase, 0.5 μl each of forward and reverse strand primers, 50 ng template DNA and the rest of the reaction was deionized water.
The annealing temperature was found to be 61℃.
The PCR product was then loaded on a 0.8% agarose gel and subjected to electrophoresis with 100 volts for 30 mins.

Learn more about our results and DBTL cycles here.

Co-Culture

Optimizing salinity for co-culture

The diatoms usually grow at higher salinities than the bacteria. We performed this experiment to test the salinity tolerance of the bacteria.
P. fluorescens was inoculated in 3 gm samples of Martian soil simulant enriched with LB media.
10 ml of the diatom’s minimal media with varying salinities was then added above the soil. Four cultures were made with salinities 30%, 15%, 7.5% and 5%. This was achieved by preparing the media in appropriate dilutions of seawater.
Growth conditions were maintained at 30°C in shaking conditions.
Growth was determined by measuring CFU in 0.1 gm samples of the soil simulant from the culture.

See our results here.
Learn more about our DBTL cycles here.

Testing the co-culture

The interaction between P. fluorescens and P. tricornutum was studied by designing a co-culture setup with conditions optimized according to growth conditions of both the organisms.
Four cultures were studied, which included one co-culture and three controls: bacterial culture, diatom culture and sterile media.
The culturing media consisted of Martian soil simulant along with the minimal media prepared in pure seawater.
For the co-culture, 3 gm of the soil simulant, enriched with 1 ml of LB media containing P. fluorescens that was grown overnight, was added to a conical flask.
Along with this, 15 ml of minimal media, with diatom cells in the exponential phase, was added to the flask.
Growth conditions were maintained at 27°C and 160 rpm.
Growth was quantified by measuring the OD of the cultures at 600 nm for bacteria and 750 nm for diatoms.

See our results here.
Learn more about our DBTL cycles here.

All the protocols that we developed for our experiments can be found here:

Bacteria

Diatoms

Co-Culture

References